Extracts of human colon carcinomas have been assayed for the presence of blood group substance A and i activities using an automated quantitative hemagglutination-inhibition system. Carcinoembryonic antigen (CEA) activity was simultaneously assayed by the zirconyl phosphate gel radioimmunoassay. Isoelectric focusing of a perchloric acid tumor extract containing all three antigen activities indicated a similar pK1 for the three antigens. However, A- or i-specific affinity gel adsorption experiments showed that only the corresponding blood group antigen activity was removed from tumor extracts. CEA activity remained essentially the same after adsorption by these gels. Significant blood group activity could not be demonstrated in preparations of highly purified CEA or in CEA from which N-acetylneuraminic acid and fucose had been removed. Conversely, CEA activity was absent from preparations of purified blood group antigens and could not be unmasked by removal of the immunodominant β-galactose of the i antigen with β-galactosidase. These data provide evidence that the antigenic sites for CEA and blood group A and i antigens are located on different molecules in colon carcinoma extracts.